Methods for cancer therapy using mutant light molecules with increased affinity to receptors

ABSTRACT

Methods and compositions are disclosed to target tumor cells with embodiments of the LIGHT proteins linked fused or conjugated to a targeting agent. These compositions bind to both human and mouse receptors with affinity sufficient to conduct preclinical and clinical trials, and with increased affinity as compared to the wild type human LIGHT protein. The targeting of embodiments of LIGHT to tumor cells reduces tumor growth and reduces metastases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/363,243, filed Jun. 5, 2014, which is a § 371 U.S. national entry ofPCT/US2012/069013, filed Dec. 11, 2012, which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/576,222, filed Dec. 15, 2011,each of which are incorporated by reference in their entireties.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under grant CA115540awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

Methods and compositions are disclosed to target tumor cells withembodiments of the LIGHT proteins linked, fused or conjugated to atargeting agent. These compositions bind to both human and mousereceptors with affinity sufficient to conduct preclinical and clinicaltrials, and with increased affinity as compared to the wild type humanLIGHT protein. The targeting of embodiments of LIGHT to tumor cellsreduces tumor growth and reduces metastases.

The paucity of activated T cells infiltrating established tumors inimmunocompetent hosts helps to explain the inability of hosts to disposeof tumors. Experiments in animal models as well as clinical studiesindicate that the immune system can recognize and kill individual tumorcells, but a host cannot generally eradicate established solid tumors.There may be several explanations for the failure of the host to respondeffectively to established tumors: 1) lack of early T cell priming dueto poor direct or indirect presentation in lymphoid tissues because ofan inadequate number of tumor cells (especially those of non-hemopoieticorigin) migrating to the lymphoid tissue; 2) inadequate numbers ofimmune cells migrating to tumor sites due to biological barriers aroundtumor tissues; 3) exhausted or short-lived activated antigen-specific Tcells that fail to combat tumor growth due to limited repertoires; 4)unresponsiveness or ignorance of T cells to tumors; 5) an inhibitorymicroenvironment or lack of stimulation inside tumors to activate theimmune system.

Clinically, an increase in the infiltration of T cells to the tumor siteis closely associated with better prognosis. There are reports thatpreventive vaccinations were effective in inducing the rejection ofinoculated tumor cells. After tumor growth has been established,however, the therapeutic vaccinations usually fail to reject tumors.Surgical reduction of a tumor does not boost the immune response totumors. Furthermore, it was reported that even the expression of astrong antigen on tumor cells was insufficient in promoting therejection of an established tumor, despite the presence of excessivenumbers of antigen-specific T cells in the lymphoid tissues. Lack of Tcells priming and/or infiltrating an established tumor is one of themajor obstacles for either natural or therapeutic approaches againstantigenic cancers. In addition, insufficient expression of costimulatorymolecules inside tumor tissues may fail to activate infiltrating T cellsand result in the anergy of tumor-reactive T cells.

The lack of early T cell priming is possibly attributed to only a fewtumor cells that migrated from solid tissue to lymphoid tissues fordirect presentation. Genetic analysis using bone marrow chimeras hasrevealed two modes of antigen presentation for priming MHC-1-restrictedCD8⁺ T cells. Direct-priming is mediated by the engagement of T cellswith the cells that synthesize the protein with antigenic epitopes,whereas cross-priming is mediated by the host antigen-presenting cellsthat take up antigens synthesized by other cells. The mechanisms bywhich tumor-specific T cells are primed has been vigorously debated andso far remains inconclusive. Understanding how and where tumor antigensare presented to T cells would help find a therapeutic action againsttumors.

LIGHT (homologous to lymphotoxin, exhibits inducible expression, andcompetes with HSV glycoprotein D for herpes virus entry mediator, areceptor expressed by T lymphocytes) is a type II transmembraneglycoprotein of the TNF ligand superfamily. LIGHT (TNFSF14) is atumor-necrosis factor (TNF) family member that interacts withLymphotoxin β Receptor (LTβR) and herpes virus entry mediator (HVEM),which are mainly expressed on stromal cells and T cells, respectively.LTβR signaling is required for the formation of organized lymphoidstructures, which can be attributed, at least in part, to its ability toinduce the expression of chemokines and adhesion molecules that attractnaive T cells and dendritic cells (DC) in lymphoid organs. Stimulationof LTβR on stromal cells by LIGHT in vivo leads to the expression ofCCL21, which attracts naive T cells in the T cell area of the spleen inthe absence of LTαβ, another ligand for LTβR. These results demonstratethat LIGHT is able to interact with LTβR to regulate CCL21 chemokineexpression. In addition, LIGHT exhibits a potent, CD28-independentco-stimulatory activity for T cell priming and expansion leading toenhanced T cell immunity against tumors and/or increased autoimmunity.Signaling via LTβR is required for the formation of organized lymphoidtissues. Lymphotoxin β Receptor (LTβR) plays an important role in theformation of lymphoid structures. LTβR is activated by two members ofthe TNF family, membrane lymphotoxina β and LIGHT. LTβR plays pivotalroles in the formation of lymph nodes (LNs) and the distinctorganization of T, B zones in secondary lymphoid organs. Signaling viaLTβR regulates the expression of chemokines and adhesion moleculeswithin secondary lymphoid organs. Chemokines and adhesion moleculescontrol the migration and positioning of DCs and lymphocytes in thespleen. Over-expression of soluble LT or TNF in non-lymphoid tissues wassufficient to promote functional lymphoid neogenesis.

LIGHT has also been called HVEM-L and LT-γ. Under the new TNFnomenclature, it is called TNFSF14. LIGHT is a 240 amino acid (aa)protein that contains a 37 aa cytoplasmic domain, a 22 aa transmembraneregion, and a 181 aa extracellular domain. Similar to other TNF ligandfamily members, LIGHT is predicted to assemble as a homotrimer. LIGHT isproduced by activated T cells and was first identified by its ability tocompete with HSV glycoprotein D for HVEM binding. LIGHT has also beenshown to bind to the Lymphotoxin B Receptor (LTβR) and the decoyreceptor (DcR3TR6).

LIGHT plays a unique role in T cell activation and the formation oflymphoid tissue. Interactions between LIGHT and LTβR restore lymphoidstructures in the spleen of LTα^(−/−) mice. In addition, theupregulation of LIGHT causes T cell activation and migration intonon-lymphoid tissues providing for the formation of lymphoid-likestructures. Conversely, LIGHT^(−/−) mice showed impaired T cellactivation and delayed cardiac rejection. Therefore, LIGHT is a potentcostimulatory molecule that also promotes the formation of lymphoidtissues to enhance local immune responses. Lack of efficient priming ofnaive T cells in draining lymphoid tissues and the inability to expandtumor-specific T cells within tumors prevent the eradication of cancer.

Micrometastases (small aggregates of cancer cells visiblemicroscopically) can become established at a very early stage in thedevelopment of heterogeneous primary tumors, and seed distal tissuesites prior to their clinical detection. For example, the detectablemetastasis in breast cancer can be observed when the primary tumor sizeis very small. Therefore, at the time of diagnosis, many cancer patientsalready have microscopic metastases, an observation that has led to thedevelopment of post-surgical adjuvant therapy for patients with solidtumors. Despite these advances, success has been limited, and optimaltreatment of metastatic disease continues to pose a significantchallenge in cancer therapy.

A variety of human and murine cancers have been proven to be antigenicand able to be recognized by T cells. Tumor-reactive T cells couldtheoretically seek out and destroy tumor antigen-positive cancer cellsand spare the surrounding healthy tissues. However, the naturallyexisting T cell responses against malignancies in human are often notsufficient to cause regression of the tumors, primary ones ormetastases. It has been reported that sporadic spontaneous, butimmunogenic tumors, avoid destruction by inducing T cell tolerance.However, the activation of tumor antigen-specific T cells may completelyprevent the development of spontaneous tumors. Thus, breaking toleranceand generating such T cells capable of rejecting tumors around the timeof treatment of the primary tumor represents a potential approach toclearing metastatic tumor cells. Because antigen-lost variants canescape under immunological pressure, immunotherapy should be applicableindependent of knowledge of specific tumor antigens.

From an immunological perspective, present clinical strategies hinderthe immune defense against malignancies and further diminish theeffectiveness of immunotherapy. Although removal of a tumor may reversetumor-induced immune suppression, surgical excision of the primary tumorbefore immunotherapy also removes the major source of antigen, which maylead to a reduction of the activation of cytotoxic T-lymphocytes (CTL)since the efficiency of priming is correlated with the tumor antigenload. In addition, current adjuvant treatments, which includechemotherapy and radiation therapy, that are meant to kill residualtumor cells may in fact impair anti-tumor immune responses by destroyingor inhibiting T cells.

Metastatic disease is the major cause of morbidity and mortality incancer. While surgery, chemotherapy, or radiation can often controlprimary tumor growth, successful eradication of disseminated metastasesremains rare. One unsolved problem is whether such response allowsincoming CTL to be educated and then exit the tumor site. Anotherunsolved problem is whether these CTL can then patrol and effectivelyeliminate spontaneously metastasized tumor cells in the periphery. Localtreatment of tumors with LIGHT generates plenty of tumor specific CTLthat exit the primary tumor and infiltrate distal tumors to completelyeradicate preexisting spontaneous metastases.

As indicated above, the naturally occurring T cell responses againstmalignancies in humans are often not sufficient to cause regression oftumors, primary ones or metastatic cells. Immunotherapy wouldpotentially elicit tumor-reactive T cells that can seek and destroydisseminated tumor antigen-positive cancer cells while sparing thesurrounding healthy tissues, but active vaccination for tumor bearinghost only shows limited benefit. Lack of well-defined antigens in mosttumors limits either active vaccination or adoptive transfer therapy.Immunotherapy that is effective even without determination of specifictumor antigens would be more applicable and more therapeuticallyfeasible. However, it is still unclear when and how to boost activeimmune responses against tumor tissues.

Naive or effector-memory T cells can leave the periphery and enter thedraining lymph nodes through an active process. It is not yet known ifsufficient number of tumor-specific CTLs recruited to the primary tumorcan survive and exit the microenvironment to patrol peripheral tissuesand eradicate disseminated metastases. In addition, a challenge indeveloping an effective immunotherapy is to devise an approach toincrease the number of, or enhance the function of, circulatingtumor-specific T cells that may detect and destroy microscopicmetastatic cells before they become clinically meaningful. The deliveryof LIGHT into the primary tumor can help generate CTL which can thenexit out of the local tumor and patrol periphery tissue to eradicatemetastases before they are clinically meaningful.

Approved breast cancer therapies include surgery, radiation,chemotherapy, (e.g. doxorubicin, paclitaxel), signaling inhibitors(e.g., Lapatinib, Neratinib), and monoclonal antibodies (e.g.Trastuzumab, Pertuzumab). Herceptin (Trastuzumab) is an approvedanti-Her2 antibody therapy for breast cancer. Her2 (human epidermalgrowth factor receptor 2 (c-erbB2 or neu), is amplified in 25-30% ofhuman breast cancers. Overexpression of Her2 is associated with poorerprognosis.

Humanized monoclonal antibody targeting Her2 employing murine antigenbinding residues on human IgG framework, was approved in 1998 by FDA formonotherapy. Overall response rates are between 11.6-35% formonotherapy.

However, there are problems with anti-Her2 antibody therapy. There arelower than desired success of treatment and large non-responsive ratewith anti-Her2/neu therapy. Anti-Her2/neu therapy requires prolongedtreatment together with chemotherapy to be effective. A majority ofpatients develop resistance and relapse within a year, and treatment cancost over $100,000 USD.

Several strategies can improve therapeutic antibody efficacy:

a. cytotoxic or immunomodulatory immunocytokines (IL-2, LIGHT etc.);

b. drug-conjugates (chemo drugs);

c. modifying Fc mediated effects, e.g., changing antibody isotypes;modifying affinity or changing Fc receptor binding; and increasinghalf-life.

Improvements to antigen-antibody binding or design may be sought by:

a. higher affinity

b. increased or decreased internalization

c. increased antibody stability

d. bi-specific, tri-specific antibodies.

In the present disclosure targeting tumors not just with wild typeLIGHT, but with various embodiments of LIGHT generates strong immunityagainst primary tumor and metastases compared to previous results withwild type LIGHT.

SUMMARY

Targeting tumor cells with embodiments of LIGHT, e.g., mutant LIGHTproteins, peptides or fragments thereof, linked, fused or conjugated toa targeting agent against a tumor reduces the growth of tumors and alsoreduces metastasis including micro-metastasis. Targeting agents includeantibodies. Further, cytokines linked to an antibody against tumorantigens are useful against micrometastasis.

LIGHT, a TNF family member is part of a complex molecular network, andis an excellent candidate for use as an immunocytokine. (FIG. 8)

LIGHT (TNFSF14) is expressed as a trimer on lymphoid tissue, immatureDCs and activated T cells.

LIGHT binds LTbR and HVEM on target cells.

LIGHT also binds soluble Decoy Receptor 3 in humans, which is secretedby many tumors.

The interplay of TNF family members controls immune regulation. Thethree pronged activity of LIGHT makes it an excellent candidate for useas an immunocytokine: LIGHT binds both LTβR and HVEM for immunemodulation; interacts with LTβR to increase chemokines and adhesionmolecules; and attracts T cells. It activates T cells and NK cellsthrough HVEM for increased immune function and tumor immunity; anddirects apoptosis of LTβR or HVEM expressing tumors. (FIG. 9)

Delivery of LIGHT to tumors using an adenoviral approach and in the formof a fusion protein was effective at reducing tumor size and controllingmetastases. (FIGS. 10, 11) (Table 1) However, production of fusionconstructs using LIGHT has been problematic. There were aggregation andlow production problems with scFV(neu)-mLIGHT fusion. Additionally, itwas difficult to show effectiveness in murine models because human LIGHTconstructs, such as anti-neu(Fab)-hLIGHT had decreased binding affinityto the mouse receptors is compared to wild type LIGHT or mouse LIGHT.

Therefore, a LIGHT molecule with increased stability and affinity wasengineered that could be used to generate a scFV(neu)-LIGHT fusionprotein or other fusion proteins that could be tested in vitro and invivo in both mouse models of disease and in humans.

Furthermore, new mutant human LIGHT molecules are disclosed which bindto the mouse receptors of LIGHT with greater affinity than wild typehuman LIGHT (hLIGHT), and have equal or greater binding to the humanreceptors of LIGHT, as compared with wild type human LIGHT. Improvementsto antibody-mediated cancer therapy and to immunotherapy for thetreatment of cancer are disclosed. “Wild type” as used herein refers toamino acid or nucleic acid sequences characteristic of sequences insource mammals, e.g., humans, mouse.

Solutions to producing improved LIGHT and improved LIGHT-targeting agentfusion constructs were achieved in part by the following steps: HumanLIGHT (hLIGHT) (“wild type”) was used as platform for engineeringbecause it is more stable than mouse LIGHT. A hLIGHT was engineered tohave equivalent or greater binding to murine LTbR and HVEM (mLTbR andmHVEM) and human LTbR and HVEM (hLTbR and hHVEM) as well as improvedexpression and stability, to ease production and increase therapeuticefficacy (FIGS. 17, 18). In that sense, engineered LIGHT molecules were“derived” from LIGHT.

hLIGHT was engineered, and clones were identified with, increased mouseLTβR and mouse HVEM binding (FIG. 21) and favorable binding to humanLTβR and human HVEM. (FIG. 23). Confirmation of binding to the mouse andhuman receptors was conducted in scFV-LIGHT fusions produced in CHOcells (FIG. 23). The human mutant LIGHT constructs were tested in vitroas a fusion protein. 7164-m4-14 LIGHT, which is one of the newhigher-affinity human mutant LIGHT molecules fused to scFV(neu), slowedthe growth of TUBO cells significantly better in comparison to the 7164antibody alone or human LIGHT alone, as measured by cell count and MTS.(FIGS. 25, 26)

The human mutant LIGHT constructs were tested in vitro and in vivo in anadenoviral construct for its potential function to stimulate host immuneresponses (FIG. 29). Human LIGHT mutant m4-14 extracellular domain wasdelivered along with a single-chain fragment (scFv) encoding an anti-neuantibody fragment (ad-neu-mutant LIGHT). Ad-neu-mutant LIGHT improvesthe CD8+ CTL response to neu both in vitro and in vivo, as compared witha construct containing the unmodified human LIGHT extracellular domain(ad-neu-human LIGHT) (FIGS. 30-32, 35). Ad-neu-mutant LIGHT alsostimulates production of increased numbers of anti-neu antibodies, ascompared with the construct containing ad-neu-human LIGHT (FIG. 33).When tested as an anti-cancer vaccine, ad-neu-mutant LIGHT is moreeffective in preventing tumors than a vaccine containing only neu (FIG.34), and has increased neu-specific cell killing (FIG. 36). LIGHT canstimulate NK cells to produce IFN via the HVEM receptor whilestimulating MEFs to produce IL-6 via LTbR. FIG. 37 demonstrates thatmutant LIGHT induced much higher IFN-γ production in Rag-1-splenocytesthan Wt LIGHT. FIG. 38 demonstrates that mutant LIGHT induced higherIL-6 production in MEF cells than Wt human LIGHT. Therefore, mutantLIGHT is a stronger stimulator than Wt LIGHT.

Inducing an immune response in tumor tissues via anantibody-human-mutant LIGHT fusion, adenoviral delivery of human mutantLIGHT, or a conjugated composition prior to surgery generates sufficientprimed antigen-specific effector T cells that exit the tumor anderadicate metastasis. An antibody specific to a cancer antigen and LIGHTthat is resistant to protease digestion (e.g., a form of mutant LIGHT inthe position 81-84 region of LIGHT) can also be administered separately.Targeting the primary tumor with TNFSF14 (LIGHT) prior to surgicalexcision is a new strategy to elicit better immune response for theeradication of spontaneous metastases. Treatment with human mutant LIGHTtreatment slows down the growth of aggressive tumors.

A composition suitable for cancer therapy includes a tumor specificantibody linked, fused or conjugated to a fragment of a human LIGHTprotein, wherein the LIGHT fragment is resistant to protease digestionin a tumor environment and is sufficient to stimulate cytotoxic Tlymphocytes against tumor cells.

A suitable composition includes a tumor specific targeting agent andmutant LIGHT amino acid sequences with mutations relative to the humanwild type sequence (FIGS. 6 and 7), or a tumor specific targeting agentlinked to a fragment of a human mutant LIGHT protein. The targetingagent and the fragment of the human mutant LIGHT protein may form afusion protein, or the fragment of the LIGHT protein may be chemicallyconjugated or linked otherwise to the targeting agent or a fragment ofthe targeting agent.

A composition includes the ability to be delivered to a tumor bysuitable methods such as direct injection, adenoviral vectors,microspheres or nanoparticles.

Any peptide fragment derived from LIGHT proteins including recombinantpeptides, synthetic peptides, recombinant LIGHT proteins, mutant LIGHTproteins, truncated LIGHT proteins, extracellular domains of LIGHT,conserved domains of LIGHT, peptide mimetics that resemble a LIGHTdomain, LIGHT proteins or peptides thereof with modified amino acids aresuitable for use in inducing immune response by linking, fusing orconjugating them a tumor specific agent, such as, for example, anantibody or a fragment thereof, provided the LIGHT fragment is capableof being stably present on a tumor cell surface and has increasedaffinity for mouse and human receptors of LIGHT.

A suitable composition includes a humanized monoclonal antibody or achimeric antibody or a heterominibody or a single chain antibody.

An antibody fragment used in conjunction with LIGHT is sufficient torecognize a tumor antigen. The fragment is sufficient to stimulatecytotoxic T lymphocytes.

A fragment of LIGHT may include about 100-150 amino acids of LIGHT. Afragment of LIGHT may have an amino acid sequence corresponding topositions about 85-240 of LIGHT. A fragment of LIGHT may also includeabout 100-150 amino acids of LIGHT. A fragment of LIGHT may include anamino acid sequence from positions about 90-240 of LIGHT. A fragment ofLIGHT may include an amino acid sequence from positions about 84-240 or83-240 or 82-240 of LIGHT.

A fragment of LIGHT may also include about 100-150 amino acids of LIGHT,provided the fragment is capable of inducing an immune response againsttumor cells. A fragment of LIGHT may include an amino acid sequence frompositions about 90-235 of LIGHT.

A fragment of LIGHT includes a protease resistant fragment. A fragmentof LIGHT may include a mutation in a protease recognition sequence EQLI(SEQ ID NO: 1).

Compositions that include the novel human mutant LIGHT extracellulardomains, are suitable for cancer treatment. A composition is disclosedwherein the LIGHT fragment includes an extracellular domain with atleast one of the following an amino acid sequences:

(SEQ ID NO: 2) QLHWRLGEMVTRLPDGPAGSWEQLIQERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYICRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV.

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 3) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLSGLSYHDGALVVTKAGYYYIYSKVQLRGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLGERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 4) RRSHEVNPAAHLTGANSSSTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLRGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 5) RRSHEVNPAAHLTGANFSLTGSGGPLLWETQLGQAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDDRLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 6) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLRGVGCPLALASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLDLRDGTRSYFGA FMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 7) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVATKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELMVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVPDERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 8) RRSHEVNPAAHLTGANFSLTGSGGPVLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKLQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRAWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 8) RRSHEVNPAAHLTGANFSLTGSGGPVLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKLQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRAWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 9) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLRGVGCPLGLASTITHGLYKRTPRYPEELELLVNQQSPCGRAPSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 10) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLRGVGCPLGLASTIAHGLYKRTPRYPEELELLVSQQSPCGRATSGSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 10) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLRGVGCPLGLASTIAHGLYKRTPRYPEELELLVSQQSPCGRATSGSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 5) RRSHEVNPAAHLTGANFSLTGSGGPLLWETQLGQAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDDRLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 11) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGFYYIYSKVQLGGVGCPLGRASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 12) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVATKAGYYYIYSKVQLGGVGCPLGLASTISHGLYKRTPRYPEELELLVSLRSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 13) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVNQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVPDERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 12) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVATKAGYYYIYSKVQLGGVGCPLGLASTISHGLYKRTPRYPEELELLVSLRSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 14) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGQAFLRGLSYHDGALVVTKAGYYYIYSKVQLRGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLARLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 15) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGQAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLANTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRMWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 16) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLRGVGCPLGLASPITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRQGDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 17) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTFTHGLYKRTPRYPEELELLVSQQSPCGRASSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 18) RSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGA FMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 19) RRSHEVNPAAHLTGANSNLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVQDERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 20) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 21) RRSHEVNPAAHLTGANSSLTGSGGPLLWEPQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLRGVGCPLGLTRTITHGLYKRTPRYPEELELLVSQQSPCGRATPSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLMDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 22) RGSHEVNPAAHLTGASSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLRGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 23) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGRASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVQDERLVRLRDGTRSYFGA FMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 24) RSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLSFLRGLSYHDGALVVTKAGYYYIYSKVQLRGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSSLGGVVHLEAGEKVVVRVLDERLVRLMDGTRSYFGA FMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 20) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFG AFMV

A composition is disclosed wherein the LIGHT fragment includes anextracellular domain with an amino acid sequence:

(SEQ ID NO: 25) QRSHEVNPAAHLTGANSSPTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSLQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRPRDGTRSYFG AFMV

Novel human LIGHT extracellular domains that binds to human and mousereceptors are listed in FIGS. 6, 7 and 28. The LIGHT mutants disclosedherein are unexpected in both their biological properties and theirefficacy. As shown in FIG. 21 (“hLIGHT mutants have increased bindingaffinity to mLTbR and mHVEM”) these new mutants not only have anincreased binding affinity for the mouse LIGHT receptors, but also havesignificantly increased binding affinity for the human LIGHT receptors,as compared with wild-type human LIGHT. In addition, these mutants haveincreased stability, as compared with the wild type human LIGHT. Inaddition to their significantly improved cross-species affinity, thesemutants are also substantially more efficacious in cell killing than thewild-type human LIGHT, as shown in FIGS. 31 and 32 (Super LIGHT canimprove CD8 CTL response to neu—in vivo killing”). It could not havebeen predicted by one of skill in the art at the time of this inventionthat mutants of LIGHT could be generated that would possess theseproperties. FIGS. 6 and 7 shows that many of the mutants have the K at214 mutated to E, which is also what is in the mouse sequence. Ofparticular interest are mutants m4-14, m4-7, and m4-16.

A method of reducing the growth of primary tumor and/or cancermetastasis, includes the steps of:

administering a pharmaceutical composition comprising a tumor-specificantibody linked to at least one of the embodiments of LIGHT, e.g., to afragment; and

reducing the growth of primary tumor and/or cancer metastasis bystimulating activation of tumor-specific T-cells against the tumor.

The antibody recognizes a surface tumor antigen and the antibody may beconjugated to the LIGHT fragment chemically or recombinantly fused orlinked otherwise to the LIGHT fragment.

The pharmaceutical composition including the antibody-LIGHT may beadministered intravenously or by other methods known to those of skillor disclosed herein.

A method of reducing the growth of primary tumor and/or cancermetastasis, includes the steps of:

(a) administering a pharmaceutical composition comprising atumor-specific antibody linked to at least one embodiment of LIGHT;

(b) introducing a nucleic acid molecule encoding the LIGHT embodimentthereof into an individual at a tumor site, wherein the LIGHT isprotease resistant; and

(c) reducing the growth of primary tumor cancer metastasis bystimulating activation of tumor-specific T-cells against the tumor.

The nucleic acid may be delivered to a pre-existing tumor site or to asite distal to a pre-existing tumor site.

A chemotherapeutic agent may also be administered during or prior to orafter an antibody-LIGHT therapy.

Radiotherapy may also be administered during or prior to or after LIGHTtherapy.

Embodiments of the antibody specific to a tumor antigen may be selectedfrom the group consisting of HER2, HER4, HERB, STEAP, c-MET, EGFR,alphafetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1,epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen(MAGE), abnormal products of ras, or p53, DcR3 and any other anti-cancerantigen.

A method of reducing the growth of primary tumor and/or cancermetastasis, includes the steps of:

(a) administering a pharmaceutical composition comprising atumor-specific antibody;

(b) introducing a nucleic acid molecule encoding a LIGHT protein or afragment thereof at a tumor site, wherein the LIGHT is proteaseresistant;

(c) expressing the LIGHT protein or a fragment thereof on the surface ofa tumor cell; and

(d) reducing the growth of the tumor and/or cancer metastasis bystimulating activation of tumor-specific T-cells against the tumor.

A chimeric protein including a peptide region that recognizes a tumorantigen and a fragment of a LIGHT protein is disclosed. The agent may bea ligand that binds a tumor surface receptor.

A composition is described including a fragment of a LIGHT protein andan agent that specifically recognizes a tumor cell.

A pharmaceutical composition includes a LIGHT peptide fragment coupledwith a tumor specific component. The tumor specific component mayinclude a ligand to a receptor in a tumor cell surface or a receptorthat recognizes a ligand on tumor cell surface.

A novel method to treat tumors (solid tumors in particular) is to createlymphoid-like microenvironments that express chemokines, adhesionmolecules, and costimulatory molecules required for priming naive Tcells and expanding activated T cells by the use of mutant LIGHTmolecules. Broader T cells are generated against tumors. Direct deliveryof antibody-LIGHT fusion or conjugates are effective against tumors andmetastasis. Tumor volume is reduced in vivo when antibody-LIGHTconjugates or fusion products are targeted to tumors as compared totumors treated with controls.

In various embodiments, the mutant human LIGHT has an amino acid changein a proteolytic site including an amino acid sequence EQLI (SEQ IDNO: 1) from positions 81-84 of native LIGHT protein. In an embodiment,the mutant LIGHT does not have the proteolytic site, an amino acidsequence EQLI (SEQ ID NO: 1) from positions 81-84 of native LIGHTprotein.

In various embodiments, the mutant human LIGHT has an amino acid changeat position 214 wherein the lysine at position 214 is changed to aglutamic acid.

The nucleic acid molecule disclosed encodes a recombinant LIGHTincluding an extracellular domain:

(SEQ ID NO: 26) QLHWRLGEMVTRLPDGPAGSWEQLIQERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV.

Cancer metastasis is reduced by stimulation of cytotoxic T-lymphocytes,and/or by stimulation of chemokines, adhesion molecules, andcostimulatory molecules for priming naive T-cells. T-cells are activatedwithin a tumor site, and may circulate in blood. Circulating T-cells arepreferably cancer specific. The T-cell generation may be CD8+ dependent.

An isolated recombinant nucleic acid includes a nucleotide sequenceencoding a protease digestion resistant mutant LIGHT. An embodiment ofthe nucleotide sequence is:

(SEQ ID NO: 27) ATGGAGGAGAGTGTCGTACGGCCCTCAGTGTTTGTGGTGGATGGACAGACCGACATCCCATTCACGAGGCTGGGACGAAGCCACCGGAGACAGTCGTGCAGTGTGGCCCGGGTGGGTCTGGGTCTCTTGCTGTTGCTGATGGGGGCTGGGCTGGCCGTCCAAGGCTGGTTCCTCCTGCAGCTGCACTGGCGTCTAGGAGAGATGGTCACCCGCCTGCCTGACGGACCTGCAGGCTCCTGGGAGCAGCTGATACAAGAGCGAAGGTCTCACGAGGTCAACCCAGCAGCGCATCTCACAGGGGCCAACTCCAGCTTGACCGGCAGCGGGGGGCCGCTGTTATGGGAGACTCAGCTGGGCCTGGCCTTCCTGAGGGGCCTCAGCTACCACGATGGGGCCCTTGTGGTCACCAAAGCTGGCTACTACTACATCTACTCCAAGGTGCAGCTGGGCGGTGTGGGCTGCCCGCTGGGCCTGGCCAGCACCATCACCCACGGCCTCTACAAGCGCACACCCCGCTACCCCGAGGAGCTGGAGCTGTTGGTCAGCCAGCAGTCACCCTGCGGACGGGCCACCAGCAGCTCCCGGGTCTGGTGGGACAGCAGCTTCCTGGGTGGTGTGGTACACCTGGAGGCTGGGGAGAAGGTGGTCGTCCGTGTGCTGGATGAACGCCTGGTTCGACTGCGTGATGGTACCCGGTCTTACTTCGGGGCTTTCATGGTG TGA,wherein the sequence encoding the protease digestion site GAGCAGCTGATA(SEQ ID NO: 28) is mutated.

Wild type human LIGHT DNA sequence (sequence encoding a protease siteEQLI (SEQ ID NO: 1) is shown in bold):

(SEQ ID NO: 29) ATGGAGGAGAGTGTCGTACGGCCCTCAGTGTTTGTGGTGGATGGACAGACCGACATCCCATTCACGAGGCTGGGACGAAGCCACCGGAGACAGTCGTGCAGTGTGGCCCGGGTGGGTCTGGGTCTCTTGCTGTTGCTGATGGGGGCTGGGCTGGCCGTCCAAGGCTGGTTCCTCCTGCAGCTGCACTGGCGTCTAGGAGAGATGGTCACCCGCCTGCCTGACGGACCTGCAGGCTCCTGGGAGCAGCTGATACAAGAGCGAAGGTCTCACGAGGTCAACCCAGCAGCGCATCTCACAGGGGCCAACTCCAGCTTGACCGGCAGCGGGGGGCCGCTGTTATGGGAGACTCAGCTGGGCCTGGCCTTCCTGAGGGGCCTCAGCTACCACGATGGGGCCCTTGTGGTCACCAAAGCTGGCTACTACTACATCTACTCCAAGGTGCAGCTGGGCGGTGTGGGCTGCCCGCTGGGCCTGGCCAGCACCATCACCCACGGCCTCTACAAGCGCACACCCCGCTACCCCGAGGAGCTGGAGCTGTTGGTCAGCCAGCAGTCACCCTGCGGACGGGCCACCAGCAGCTCCCGGGTCTGGTGGGACAGCAGCTTCCTGGGTGGTGTGGTACACCTGGAGGCTGGGGAGAAGGTGGTCGTCCGTGTGCTGGATGAACGCCTGGTTCGACTGCGTGATGGTACCCGGTCTTACTTCGGGGCTTTCATGG TGTGA-3′.

Native human LIGHT amino acid sequence (protease digestion site is boldunderlined):

(SEQ ID NO: 30) MEESVVRPSVFVVDGQTDIPFTRLGRSHRRQSCSVARVGLGLLLLLMGAGLAVQGWFLLQLHWRLGEMVTRLPDGPAGSW EQLI QERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV

One aspect of a mutant human LIGHT amino acid sequence (EQLI (SEQ IDNO: 1) is absent, indicated by dots):

(SEQ ID NO: 31) MEESVVRPSVFVVDGQTDIPFTRLGRSHRRQSCSVARVGLGLLLLLMGAGLAVQGWFLLQLHWRLGEMVTRLPDGPAGSW . . . QERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGA FMV.

Codon optimized nucleotide sequence for mouse mutant LIGHT, starting ATGis highlighted in bold:

(SEQ ID NO: 32) GGGCGAATTGGGTACCGGATCCGCCACCATGGAGAGCGTGGTGCAGCCCAGCGTGTTCGT 1---------+---------+---------+---------+---------+---------+GGTGGACGGCCAGACCGACATCCCCTTCAGGAGGCTGGAGCAGAACCACAGGCGGAGGAG 61---------+---------+---------+---------+---------+---------+ATGTGGCACCGTGCAGGTGTCCCTGGCCCTGGTGCTGCTGCTGGGCGCTGGCCTGGCCAC 121---------+---------+---------+--------+---------+---------+CCAGGGCTGGTTTCTGCTGAGGCTGCACCAGAGGCTGGGCGACATCGTGGCCCACCTGCC 181---------+---------+---------+--------+---------+---------+CGATGGCGGCAAGGGCAGCTGGCAGGACCAGAGGAGCCACCAGGCCAACCCTGCCGCCCA 241---------+---------+---------+--------+---------+---------+CCTGACAGGCGCCAACGCCAGCCTGATCGGCATCGGCGGACCCCTGCTGTGGGAGACCAG 301---------+---------+---------+--------+---------+---------+GCTGGGCCTGGCTTTCCTGAGGGGCCTGACCTACCACGACGGCGCCCTGGTGACCATGGA 361---------+---------+---------+--------+---------+---------+GCCCGGCTACTACTACGTGTACAGCAAGGTGCAGCTGTCCGGAGTGGGCTGCCCTCAGGG 421---------+---------+---------+--------+---------+---------+CCTGGCCAACGGCCTGCCCATCACCCACGGCCTGTACAAGAGGACCAGCAGATACCCCAA 481---------+---------+---------+--------+---------+---------+GGAGCTGGAGCTGCTGGTCTCCAGGCGGAGCCCCTGTGGCAGGGCCAACAGCAGCCGAGT 591---------+---------+---------+--------+---------+---------+GTGGTGGGACAGCAGCTTCCTGGGCGGCGTGGTGCACCTGGAGGCCGGCGAGGAGGTGGT 601---------+---------+---------+--------+---------+---------+GGTGAGGGTGCCCGGCAACAGGCTGGTGAGGCCCAGGGACGGCACCAGGAGCTACTTCGG 661---------+---------+---------+--------+---------+---------+ CGCCTTCATGGTGTGATGAGCGGCCGCGAGCTCCAGCTTTTGTTCCC721---------+---------+---------+---------+-------GCGGAAGTACCACACTACTCGCCGGCGCTCGAGGTCGAAAACAAGGG

Codon optimized nucleotide sequence for human mutant LIGHT, starting ATGis highlighted in bold.

(SEQ ID NO: 33) GAATTCGAGCTCGGTACCCGACACGGTACCGGATCCGCCACCATGGAGGAGAGCGTTGTGAGGCCCAGCGTGTTCGTGGTGGACGGCCAGACCGACATCCCCTTCACCCGGCTGGGCCGGAGCCACCGGAGGCAGAGCTGCTCCGTGGCCAGAGTGGGGCTGGGCCTGCTGCTCCTGCTGATGGGAGCCGGCCTGGCCGTGCAGGGCTGGTTCCTGCTGCAGCTGCACTGGCGGCTGGGCGAGATGGTGACCCGGCTGCCCGATGGCCCTGCCGGCAGCTGGCAGGAGCGGCGGAGCCACGAGGTGAACCCTGCCGCCCACCTGACCGGCGCCAACAGCAGCCTGACCGGCAGCGGCGGACCCCTGCTGTGGGAGACCCAGCTGGGCCTGGCCTTCCTGAGGGGCCTGAGCTACCACGACGGCGCCCTGGTGGTGACCAAGGCCGGCTACTACTACATCTACAGCAAGGTGCAGCTGGGCGGAGTGGGCTGCCCTCTGGGGCTGGCCAGCACCATCACCCACGGCCTGTACAAGCGGACCCCCAGATACCCCGAGGAGCTGGAGCTGCTGGTGTCCCAGCAGAGCCCCTGTGGCAGGGCCACCTCCAGCAGCCGGGTGTGGTGGGACAGCAGCTTCCTGGGCGGCGTGGTGCACCTGGAGGCCGGCGAGAAAGTGGTTGTGAGGGTGCTGGACGAGCGGCTTGTGAGGCTGAGGGACGGCACCCGGAGCTACTTCGGCGCCTTCATGGTGTGATGAGCGGCCGCGAGCTCGTCTCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTG

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematic illustrations of the AG 104A tumor specificheterodireic constructs. Human C.kappa. was fused via the flexible upperhinge region of human IgG3 to the C-terminus of a scFv-fragment that wasderived from a cancer antigen.

FIG. 2 demonstrates that Adv-mmlit inhibits neu+N202 tumor growth. About8×10⁵ N202 1A cells were injected (i.c.). Intratumoral injections ofabout 2×10¹⁰ vp adv-lacz or adv-mmlit were performed at day 18 and day20. The size of tumor was monitored twice a week.

FIG. 3 shows the scFv-LIGHT fusion protein structure design. FIG. 3discloses the “SL: Short linker” and “LL: Long linker” sequences as SEQID NOS 41 and 42, respectively.

FIG. 4 shows suppression of tumor growth after anti-Her2 and Ad-LIGHTtreatment that 10⁶ TUBO tumor cells were inoculated to BABL/c mice s.c.10¹⁰ VP of Ad-LIGHT or Ad-LacZ was injected intratumorally at Day 18after tumor inoculation. 50 μg anti-Her 2 antibody or isotype IgG wasinjected i.p. at Day 18 and 25 after tumor inoculation. Tumor growth wasmonitored at indicated time points. All of the treated groups havesignificant difference compared with isotype IgG group after Day 21.Ad-LIGHT and anti-Her2 combination treatment group has significantsynergistic difference compared with either Ad-LIGHT alone or anti-Her2alone group after Day 25. Statistic analysis was performed with two-tailstudent's test. Data shown were means+SEM. p<0.05 was regarded assignificant difference.

FIG. 5 shows suppression of tumor growth after anti-Her2 treatment. 10⁶TUBO tumor cells were inoculated to BALB/c mice s.c. 100 μg anti-Her 2antibody or isotype IgG was injected i.p. at Day 10 and 17 after tumorinoculation. Tumor growth was monitored at indicated time points. Tumorregrew in three out of five mice treated with anti-Her 2.

FIG. 6 shows the protein sequences of the new human light mutants. Themutated amino acids are noted in bold, italics and underlining. Many ofthe mutants relative to human wild type LIGHT have K mutated to E atposition 214—similar to the mouse sequence. FIG. 6 discloses SEQ ID NOS43-44, 6-8, 8-10, 10, 4, 3, 5, 5, 11-13, 12, 14-17, 20, 19-23, 45, 20,and 25, respectively, in order of appearance.

FIG. 7 lists amino acid sequences for novel human LIGHT mutants. Ofparticular interest are m4-14, m4-7 and m4-16. FIG. 7 discloses SEQ IDNOS 6-8, 8-10, 10, 4, 3, 5, 5, 11-13, 12, 14-17, 20, 19-23, 45, 20, and25, respectively, in order of appearance.

FIG. 8 illustrates the LIGHT network in humans. LIGHT (TNFSF14) isexpressed as a trimer on lymphoid tissue, immature DCs and activated Tcells, and binds LTbR and HVEM on target cells. LIGHT also binds solubleDecoy Receptor 3 (DcR3) in humans, which is secreted by a number ofcancers, including gastrointestinal, bone, lung, and soft tissue tumors.The interplay of TNF family members as shown controls immune regulation.

FIG. 9 illustrates the 3-pronged activity of LIGHT. LIGHT binds bothLTβR and HVEM for immune modulation, and interactions with LTβR increasechemokines and adhesion molecules and attract T cells. LIGHT activates Tcells and NK cells through HVEM for increased immune function and tumorimmunity. Finally, LIGHT can direct apoptosis of LTβR or HVEM expressingtumors.

FIG. 10 shows that expression of LIGHT near mammalian tumors in vivocontrols metastases. (C) 4T1, a normally poorly immunogenic mammarycarcinoma cell line, mimics breast cancer when injected into the mammaryfat pad of mice. It can metastasize to various organs, including lungs.In vitro-cultured 4T1 mammary carcinoma (1×10⁵ cells) were infected withAd-LIGHT or Ad-control (2×10⁸ PFU/ml) for 24 h and then 1×10⁸ cells wereinjected s.c. into the flank of BALB/c mice. Tumor growth was monitored(A) until mice were sacrificed on day 35 posttumor inoculation foranalysis of (B) lung metastases with colonogenic assay.

FIG. 11 shows that Ad-LIGHT treatment eradicates established metastases.A, 4T1 mammary carcinoma cells injected s.c. into the flank of BALB/cmice were treated intratumorally with 1×10⁹ PFU Ad-LIGHT (black) orAdcontrol (white) on days 14 and 17 posttumor inoculation. One group ofmice was treated with surgery alone on day 14 posttumor challenge(dotted). Other 4T1 tumor-bearing mice were treated with Ad-LIGHT in thesame way, with the addition of CD8 depletion by anti-CD8 Ab (YTS.169.4.2), starting day 14 after primary tumor inoculation (gray).Anti-CD8 Ab was given to mice i.p., 125 μg/mouse once every week untilthe mice were sacrificed for analysis. More than 90% of CD8+ T cellswere depleted by this regimen, as confirmed by FACS staining ofperipheral blood. Except for the mice that were treated with surgeryalone, the primary tumors (about 150 mm3) on other mice were surgicallyresected on day 24 and mice were sacrificed for analysis of lungmetastases with colonogenic assay on day 35. Data are a pool of multipleindependent experiments.

FIG. 12 shows models of a T-cell receptor with the CDR1, CDR2, and CDR3regions highlighted. FIG. 12 discloses SEQ ID NO: 35.

FIG. 13 shows peptide specificity of CDR mutants, QL9/Ld binding andpeptide-Ld specificity of various CDR mutants. Peptide selectivity ofyeast-displayed 2C mutants selected on QL9/Ld. Mutants in (A) CDR3a, (B)CDR3b, (C) CDR1b, and (D) CDR2a were assayed with (0.4 mM) the indicatedpeptide/Ld/Ig dimer, or the secondary reagent (PE) alone. MCMV,YPHFMPTNL (SEQ ID NO: 34); QL9, QLSPFPFDL (SEQ ID NO: 35); QL9 variantscontained single amino acid substitutions at position 5 (wild-type, F).Yeast cells were assayed by flow cytometry for binding of the pep-Ldcomplexes (MFU, mean fluorescence units).

FIG. 14 is a ribbon diagram of the mutant Ly49C-H-2Kb complex, in whichthe crystallographic Ly49C dimer (arrow) crosslinks two MHC class Imolecules.

FIG. 15 illustrates the “domino” effect of peptides on pepMHC bindingand specificity. There is a network of hydrogen bonding interactionsthat cascade to the Ly49C contact region. Different peptides may alterMHC conformation and change Ly49C binding.

FIG. 16 illustrates crystallization of 2B4-CD48, and the mouse NK cellimmune synapse. The structure of the Ly49C-H-2Kb complex (1P4L) does notinclude the 70-residue stalk regions that connect the Ly49C homodimer tothe NK cell membrane.

FIGS. 17 and 18 illustrate the engineering of the new human LIGHTmutants using Yeast Display (YD). Human LIGHT was fused to matingadhesion receptor Aga2. Fluorescent epitope tags were used fornormalization, and equilibrium, kinetic and thermal stability analysiswas assessed by flow cytometry.

FIG. 19 shows how the human LIGHT library was generated for use in YD.Human LIGHT was subjected to variable error-rate error-prone PCR, whichgenerated 4.5×10⁷ clones. Selection was performed using the mouse andhuman LIGHT receptors mLTβR, mHVEM and hLTβR, hHVEM, respectively.

FIG. 20 shows the binding properties of isolated mutants (constructs)when tested against the mouse and human LTβR and HVEM.

FIG. 21 shows the increased binding affinity of the human LIGHT mutantsto mLTβR and mHVEM.

FIG. 22 illustrates an scFV (neu)-LIGHT fusion protein. The fusionprotein was generated with a c-terminal streptavidin tag II for enhanceddetection by western blot, flow cytometry and ELISA, and for highspecificity, one-step purification.

FIG. 23 (A-D) shows favorable binding of 4 mutant human LIGHT clones formLTβR, mHVEM and hLTβR, hHVEM.

FIG. 24 shows that scFV and LIGHT bind their respective ligands whenproduced as an scFV-LIGHT fusion protein.

FIGS. 25 and 26 show that scFV(neu)-LIGHT fusion protein decreasesgrowth of TUBO cells in culture. TUBO is a cloned cell line generatedfrom a spontaneous mammary gland tumor from a BALB-neuT mouse and highlyexpresses HER-2 protein on the cell membrane. (A) TUBO cells werecultured and treated with 5 ug/mL protein, and assessed for growth afterfour days. (B) The fusion protein 7164m4-14 LIGHT, generated from ananti-neu single chain antibody and the mutant human LIGHT m4-14,significantly decreased growth of TUBO cells as compared to theantibody-treated or untreated cells.

FIG. 27 shows that cell death caused by 7164m4-14 LIGHT is mediated viathe interaction between LIGHT and LTβR.

FIG. 28 shows nucleic acid sequence of the human LIGHT extracellulardomain (SEQ ID NO: 46) and the human LIGHT m4-14 (SEQ ID NO: 47) nucleicacid sequence.

FIG. 29 are diagrams of scFV-LIGHT (85-239) fusion proteins.

FIG. 30 shows that superLIGHT, an adenoviral construct that links a neusequence via an IRES to the human mutant LIGHT m4-14, improves CD8-CTLresponse to new ICS. WT B/C mice were immunized with 5×10⁸ IFUad-neu-human LIGHT or ad-neu mutant LIGHT, and after 10 days, thesplenocytes were made into singe cells and stimulated with neu-HISprotein or peptide RatP66, or left unstimulated. ICS was performed todetect IFN-gamma positive CD8 cells.

FIGS. 31 and 32 show superLIGHT (m4-14 mutant) improves CD8 CTL responseto neu in vivo killing. WT B/C mice were immunized with (B) 5×10⁸ IFUad-neu-human LIGHT or (C) ad-neu mutant LIGHT. After 10 days, (A) naïvesplenocytes were labeled with 0.5 uM and 5 uM CFSE and the 0.5 uM cellswere also loaded with peptide Ratp66, equal numbers of the cells weremixed together and tail vein injected into naïve mice or immunized mice.After 16 hours, the spleen and draining lymph nodes (LN) were analysizedfor CFSE positive cells.

FIG. 33 shows super LIGHT induces higher anti-neu antibody after NAvaccine. Naïve mice (4 mice in each group) were immunized twice at 14day intervals with PcDNA-neu or PcDNA-neu-LIGHT (human or mutant) byhydrodynamic injection. Seven days after the second immunization, mouseserum was collected and anti-neu antibody was tested using cellularELISA.

FIG. 34 shows improved efficiency of new super LIGHT DNA vaccine in 10of tumor free mice (in vivo). Naïve mice (4 mice in each group) wereimmunized with PcDNA-neu or PcDNA-neu-LIGHT (mutant) by hydrodynamicinjection. Forty four days later, the immunized mice were inoculatedwith 5*10⁵ TUBO cells. Tumor free mice were sacrificed and analyzedafter another 40 days.

FIGS. 35 and 36 shows that super LIGHT improves neu-specific killingcompound to hLIGHT, Naïve Balb/c mice was vaccinated subcutaneously withseveral different doses of adenovirus. After eleven days, ICS isperformed as the following: naïve splenocytes were labeled with 0.5 uMor 5 uM CFSE, and the CFSE low cells were loaded with her2/neu peptide,then equal numbers of CFSE low and high cells were injected into theimmunized mice by tail vein, 20 hours later, the CFSE positive cells inthe spleen of vaccinated mice were analyzed by FACS.

FIG. 37 demonstrates that LIGHT induced IFN-γ in Rag-1-Splenocytes.

FIG. 38 demonstrates that LIGHT induced IL-6 in MEF cells.

DETAILED DESCRIPTION

Metastatic disease is a major cause of mortality among cancer patients.Initial dormancy of metastasis or small primary tumors may be attributedto the insufficient levels of antigens available to prime CD8+ T cells.Therapeutic methods that utilize LIGHT and mutants of human LIGHT caneffectively target CD8+ T cells. Combining LIGHT or human mutant LIGHTwith an antibody that recognizes an antigen expressed by tumor cells(antibody-LIGHT) can specifically and effectively target migrant tumorcells after such Antibody-LIGHT is introduced systemically byintravenous (i.v.) injection.

As an example, in a mouse model, a high-affinity monoclonal antibodyagainst tumor cells accumulates inside tumors in vivo with highconcentration after intravenous injection. The heterominibody LIGHT (byconjugation or genetic linkage) allows LIGHT to be specificallydelivered into tumor tissue at various distal sites after its systemicintroduction.

A LIGHT fusion protein (e.g., antibody-LIGHT couple) selectivelyaccumulates inside tumor tissues and specifically binds to tumors invitro.

Therapeutic methods that utilize an antibody recognizing an antigenexpressed by tumor cells coupled with LIGHT (antibody-LIGHT) aredesigned to specifically and effectively target migrant tumor cellsafter the Antibody-LIGHT is introduced systemically by intravenousinjection. Any tumor antigen that is expressed on the surface of thetumor cell or is capable of being recognized by a tumor-specificantibody is suitable to be coupled with LIGHT or a functional fragmentthereof.

Local delivery of a protease resistant LIGHT (e.g., a mutant LIGHT or anextracellular domain of LIGHT) enhances direct presentation of tumorantigens to antigen-specific T cells and prevents anergy of infiltratedT cells within the tumor microenvironment. In addition, LIGHT mayenhance tumor apoptosis in vivo.

Successful eradication of metastasis by currently available cancertreatments remains rare. Generating immune responses in primary tumortissues prior to surgical resection produces tumor-specific effector Tcells sufficient to eradicate distant metastases. Priming oftumor-specific CD8⁺ T cells, for example by antibody-LIGHT delivery inthe primary tumor promotes subsequent exit of cytotoxic T lymphocytes(CTL) that home to distal tumors. Targeting primary tumor prior tosurgical excision elicits immune-mediated eradication of spontaneousmetastasis.

Metastasis is often a fatal step in the progression of solidmalignancies. Disseminated metastatic tumor cells can remain dormant andclinically undetectable for months or even years following surgicalresection of the primary tumor, leading to subsequent clinical diseaserecurrence. Immunotherapeutic strategies are suitable to eliminate thismicrometastatic disease. As an example, delivery of antibody-LIGHT intothe primary tumor reduces the formation of metastasis and rejects theestablished metastasis in peripheral tissues. For example, directdelivery of LIGHT in the form of an antibody-LIGHT fusion protein totumors (e.g., primary tumor) generates sufficient number ofeffector/memory T cells from the tumor tissues that move to a distalsite, leading to an overall increase in the intensity of the immuneresponse, greater inflammatory cytokine production, and the eradicationof spontaneous metastasis. Immunotherapy using primary tumor tissuesaimed to provoke and sustain a tumor specific immune response in thepresence of endogenous tumor antigens generates the necessary CTL toclear already disseminated tumor cells.

In the presence of LIGHT on the surface of a tumor, CTLs are efficientlyprimed and subsequently circulate to infiltrate LIGHT-negative distaltumors. Without the benefits of LIGHT being present in the primarytumor, few activated T cells are expected at a secondary tumor site. Itis likely that these effector/memory T cells generated in the localtumor site in the presence of LIGHT are able to exit the tumor andpatrol the periphery and identify metastatic tumor cells. Chemokinereceptor (CCR7) has been recently shown to be a key molecule for T cellsto exit the peripheral tissues, including the inflammatory site, andtraffic to the draining LN. The 2C T cells exiting LIGHT-expressingtumors may be controlled by CCR7.

For example, an extracellular domain of LIGHT molecule can berecombinantly expressed such that either the recombinant form does nothave the proteolytic site all together or has one or more amino acidchanges that renders the recombinant form protease digestion resistant(mutant LIGHT). In addition, the extracellular domain or a functionalequivalent derivative of the extracellular domain of LIGHT can be linkedto a tether or linker or spacer sequence to anchor the extracellulardomain in the membrane of tumor cells. FIGS. 2-3 illustrate some aspectsof an antibody-LIGHT fusion or conjugation.

The extracellular domain of LIGHT refers to a form of the LIGHTpolypeptide which is essentially free of the transmembrane andcytoplasmic domains. The extracellular domain of LIGHT has less than 1%of such transmembrane and/or cytoplasmic domains and preferably, willhave less than 0.5% of such domains. It is to be understood that anytransmembrane domains identified for the LIGHT polypeptides areidentified pursuant to criteria routinely employed in the art foridentifying that type of hydrophobic domain. The exact boundaries of atransmembrane domain may vary but most likely by no more than about 2-5amino acids at either end of the domain as initially identified herein.An extracellular domain of a LIGHT polypeptide may contain from about 5or fewer amino acids on either side of the transmembranedomain/extracellular domain boundary as identified herein.

Suitable LIGHT protein, protein and peptide fragments thereof, includefor example, amino acid positions 1-240 of LIGHT without one or more ofthe amino acids representing the proteolytic site EQLI (81-84) (SEQ IDNO: 1); amino acid positions 1-240 of LIGHT with one or more of theamino acids representing the proteolytic site EQLI (81-84) (SEQ IDNO: 1) is mutated or otherwise inactivate; 82-240 of LIGHT; 83-240 ofLIGHT; 84-240 of LIGHT; 85-240 of LIGHT; 90-240 of LIGHT; 95-240 ofLIGHT; 100-240 of LIGHT; 85-235 of LIGHT; 85-230 of LIGHT; 85-225 ofLIGHT; 85-220 of LIGHT; 85-215 of LIGHT; 85-200 of LIGHT; LIGHT fragmentwithout the intracellular and membrane domain; and any fragment that isabout 100-150 amino acids in length of LIGHT that is resistant toprotease digestion.

“Antibody-LIGHT” refer to an antibody or a fragment thereof specificagainst a tumor antigen, which is either fused or conjugated to afragment of LIGHT protein that is sufficient to trigger an immuneresponse against tumor cells and is capable of being stably present on atumor cell surface by being resistant to protease digestion compared toa native LIGHT protein.

As used herein, the term “LIGHT” in an antibody-LIGHT couple refers toeither an extracellular domain of LIGHT that does not contain a proteaserecognition sequence, or a mutant LIGHT wherein the protease site (EQLI)(SEQ ID NO: 1) is inactivated by entire deletion or a mutation at one ormore amino acids that render the protease site insensitive or inactiveor a truncated form of LIGHT that is resistant to protease digestion andcapable of stimulating T-cells. LIGHT may also refer to a novel sequencein FIGS. 6 and 7.

“Mutant LIGHT” refers to a LIGHT protein or a LIGHT-derived peptide thatis resistant to proteolytic cleavage, capable of being stably expressedin the surface of tumor cells, and exhibits increased activation oftumor specific T-cells, compared to normal or native LIGHT protein. The“mutant LIGHT” relates to a LIGHT protein or LIGHT protein-derivedpeptides or fragments that are resistant to protease digestion orotherwise are capable of being stably expressed on the surface of cellsincluding tumor cells because of a mutation that renders the proteolyticsite EQLI (SEQ ID NO: 1) inactive. There are several ways to generatemutant LIGHT. For example, the protease site (e.g., EQLI) (SEQ ID NO: 1)can be mutated either to remove the protease site in toto or to renderthe site resistant to protease digestion by changing (e.g., insertion,deletion, substitution) one or more amino acids at the protease site.

“Truncated LIGHT” protein refers to a LIGHT fragment that is not fulllength when compared to a native LIGHT, is resistant to proteasedigestion and is capable of stimulating T-cells against tumor cells. Forexample, the extracellular domain of LIGHT (about 85-240) is a suitabletruncated LIGHT. Truncated LIGHT includes fragments/derivatives of LIGHTprotein that are resistant to protease digestion thereby exhibiting theability to be present on the cell surface for an extended period of timecompared to native LIGHT protein.

To generate protease resistant LIGHT protein (e.g., mutant LIGHT) orfragments or LIGHT protein or LIGHT peptides with the protease siteinactivated, for example, the amino acid glutamic acid (E), can bedeleted or substituted within the protease recognition sequence EQLI(SEQ ID NO: 1). Similarly, the amino acid glutamine (Q) is deleted orsubstituted with another amino acid within the protease recognitionsequence EQLI (SEQ ID NO: 1). Similarly, amino acid L or I can bedeleted or substituted with other amino acids. Protease resistant aminoacid analogs can also be used to generate synthetic LIGHT fragments thatprotease resistant. For example, using the incorporation of B-aminoacids into peptides decreases proteolysis and can be used to substitutethe protease sensitive site EQLI (SEQ ID NO: 1). Rational incorporationof B-amino acids within the protease site and near the protease site canbe performed and the resulting mutants tested for protease resistance. Avariety of techniques including site directed mutagenesis can be used togenerate LIGHT fragments that are resistant to protease digestion.

The term “inactivated” means that the LIGHT protein or its fragmentsthereof is resistant to protease digestion in a tumor environmentbecause the protease recognition site has been selectively silencedeither by mutation in one or more amino acids or by deletion of EQLI(SEQ ID NO: 1) or by substitution of one or more amino acids with a- orB-amino acids or by any suitable way.

The term “resistant” means that the LIGHT protein or its fragmentsthereof is not sensitive to protease digestion in a tumor environmentbecause the protease recognition site has been inactivated/mutatedeither by mutation in one or more amino acids or by deletion of EQLI(SEQ ID NO: 1) or by substitution of one or more amino acids with a- orB-amino acids or by any suitable way.

The term “tumor environment” refers to the presence and expression andactivity of cellular proteases including extracellular proteases thatmay co-operatively influence matrix degradation and tumor cell invasionthrough proteolytic cascades, with individual proteases having distinctroles in tumor growth, invasion, migration, angiogenesis, metastasis andexpansion of tumors.

“Ad-LIGHT” or “Ad-mutant LIGHT” refers to recombinant adenoviral vectorsystem that contains mutant LIGHT encoding nucleic acids and is suitablefor delivering the nucleic acid sequences to a tumor site or capable ofinfecting tumor cells. “Metastasis or metastases” refers to the processby which cancer spreads from the location at which the cancer initiatedas a tumor to one or more distant locations in the body by migration ofone or more cancerous cells. These terms also include micro-metastasiswherein the formation of tumors at distal locations corresponds to smallaggregates of cancer cells that are visible microscopically. These termsalso refer to the secondary cancerous growth resulting from the spreadof the primary tumor from the original location.

“Reducing or controlling metastasis” refers to a reduction in the numberof metastatic tumor sites as compared to a control.

“Adoptive transfer” refers to the transfer of T cells into recipients.

“Tumor site” means a location in vivo or ex vivo that contains or issuspected of containing tumor cells. Tumor site includes solid tumorsand also the locations that are adjacent or immediately near a tumorgrowth.

“Tumor-specific” refers to antibody or any other ligand/receptor thatshows preference to tumor cells over normal cells. For example, anantibody targeted to an antigen present on tumor cells is consideredtumor-specific. A tumor-specific antibody may also bind to a normal cellif the target antigen is present, albeit to a lesser degree.

As used herein, the term “administration” refers to systemic and/orlocal administration. The term “systemic administration” refers tonon-localized administration such that an administered substance mayaffect several organs or tissues throughout the body or such that anadministered substance may traverse several organs or tissues throughoutthe body in reaching a target site. For example, administration into asubject's circulation may result in expression of a therapeutic productfrom an administered vector in more than one tissue or organ, or mayresult in expression of a therapeutic product from an administeredvector at a specific site, e.g., due to natural tropism or operablelinkage of tissue-specific promoter elements. One of skill in the artwould understand that various forms of administration are encompassed bysystemic administration, including those forms of administrationencompassed by parenteral administration such as intravenous,intramuscular, intraperitoneal, and subcutaneous administration. In someembodiments, systemic administration can be used to elicit a systemiceffect associated with treatment of a local or systemic disease orcondition. A systemic effect may be desirable for a local disease orcondition, for example, to prevent spread of said disease or condition.The term “local administration” refers to administration at or near aspecific site. One of skill in the art would understand that variousforms of administration are encompassed by local administration, such asdirect injection into or near a specific site. In some embodiments,local administration is associated with treatment of a disease orcondition where a local effect is desired (e.g. administration to thelung for the treatment of lung cancer). A local effect may be desired inassociation with either local or systemic diseases or conditions. Alocal effect may be desired in association with a systemic disease orcondition to treat a local aspect of a systemic disease or condition.

An “effective amount” of LIGHT, LIGHT polypeptide or peptide, or afragment thereof, LIGHT fusion products, or LIGHT conjugates, and thelike, refers to an amount sufficient to carry out a specifically statedpurpose. An “effective amount” may be determined empirically and in aroutine manner, in relation to the stated purpose. For example, asuitable purpose for an antibody-LIGHT construct is reducing tumor sizeor growth and/or reduce metastases.

The term “therapeutically effective amount” refers to an amount ofLIGHT, LIGHT polypeptide or peptide or a fragment thereof, LIGHT fusionproducts or conjugates, effective to treat a specific disease ordisorder in a subject or mammal. In the case of cancer, thetherapeutically effective amount of the compositions disclosed hereinmay reduce the number of cancer cells; reduce the tumor size; inhibit(i.e., slow and/or stop) cancer cell infiltration into peripheralorgans; inhibit (i.e., slow and/or stop) tumor metastasis; inhibit tumorgrowth; and/or relieve one or more of the symptoms associated with thecancer.

The term “antibody” covers, for example, monoclonal antibodies,polyclonal antibodies, single chain antibodies, fragments of antibodies(see below) as long as they exhibit the desired biological orimmunological activity. The term “immunoglobulin” (Ig) is usedinterchangeable with antibody herein. The antibodies may specificallytarget a tumor antigen, e.g., surface tumor antigen such as for exampleHer2/neu and CD20.

An “isolated antibody” is one which has been identified and separatedand/or recovered from a component of its natural environment. Theantibody is purified to greater than 95% by weight of antibody asdetermined by the Lowry method, and more than 99% by weight.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies of the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site or an epitope. For example, themonoclonal antibodies may be prepared by the hybridoma methodology firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made usingrecombinant DNA methods in bacterial, eukaryotic animal or plant cells(see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” mayalso be isolated from phage antibody libraries using the techniquesdescribed in Clackson et al., Nature, 352:624-628 (1991) and Marks etal., J. Mol. Biol., 222:581-597 (1991), for example.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate, andhuman constant region sequences.

“Antibody fragments” include a portion of an intact antibody, forexample the antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab¹, F(ab¹)₂, single chainF_(v) and F_(v) fragments; diabodies; linear antibodies (see U.S. Pat.No. 5,641,870; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies mayinclude residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody includes substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also includes at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include carcinoma, lymphoma,blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung andsquamous carcinoma of the lung, cancer of the peritoneum, hepatocellularcancer, gastric or stomach cancer including gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, cancer of the urinary tract, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, melanoma, multiple myeloma and B-celllymphoma, brain, as well as head and neck cancer, and associatedmetastases.

Suitable surface tumor antigens that can be targeted using aantibody-LIGHT fusion or conjugate includes epidermal growth factorreceptor family (EGFR) including HER1, HER2, HER4, and HER8 (Nam, N. H.,& Parang, K. (2003), Current targets for anti-cancer drug discovery.Current Drug Targets, 4(2), 159-179), STEAP (six-transmembraneepithelial antigen of the prostate; Hubert et al., STEAP: aprostate-specific cell-surface antigen highly expressed in humanprostate tumors, Proc Natl Acad Sci USA. 1999; 96(25):14523-8.), CD55(Hsu et al., Generation and characterization of monoclonal antibodiesdirected against the surface antigens of cervical cancer cells, HybridHybridomics. 2004; 23(2):121-5.). Other suitable antibodies includeRituximab (Rituxan™, a chimeric anti-CD20 antibody), Campath-1H(anti-CD52 antibody), and any cancer specific cell-surface antigens. Thefollowing is an exemplary list of approved monoclonal antibody drugsagainst specific cancer types that are suitable for use with LIGHTprotein: Alemtuzumab (Campath™) for chronic lymphocytic leukemia;Bevacizumab (Avastin™) for colon cancer and Lung cancer; Cetuximab(Erbitux™) for colon cancer and head and neck cancer; Gemtuzumab(Mylotarg™) for Acute myelogenous leukemia; Ibritumomab (Zevalin™) fornon-Hodgkin's lymphoma; Panitumumab (Vectibix™) for colon cancer;Rituximab (Rituxan™) for Non-Hodgkin's lymphoma; Tositumomab (Bexxar™)for non-Hodgkin's lymphoma; and Trastuzumab (Herceptin™) for breastcancer.

EXAMPLES

The following examples are for illustrative purposes only and are notintended to limit the scope of the disclosure.

Example 1—Coupling or Conjugating Light to a Tumor Targeting Agent

To enable delivery of a mutant LIGHT delivery system or an equivalentdelivery system, mutant LIGHT can be coupled or conjugated to a tumortargeting agent such as a tumor specific antibody. For example, a tumorspecific antibody conjugated to LIGHT or mutant LIGHT can be used toselectively deliver the fusion protein to the tumor site. In addition, atumor specific antibody can be designed to be coupled with a viraldelivery system or a liposome vehicle delivery system. The deliveryvehicle expressing the mutant LIGHT and harboring the tumor targetingagent will first target the specific tumor cell and then transform thetumor cell to express mutant LIGHT on the surface of the cell. Thistargeted mutant LIGHT expression on the surface of the tumor cells willinduce chemokines on stromal cells surrounding the tumor to attract andinitiate priming of T-cells. Such treatments are suitable for alltumors, including solid tumors. 4T1, MC38, B16, and mastocytoma weretreated with Ad-LIGHT and showed a reduction of primary and/or secondarytumors. Therefore, antibody-LIGHT can be used to target various tumors,especially metastases that form as a result of cells of the primarytumor migrating to distant sites. For example, through systemicinjection, anti-her2/neu antibody with LIGHT can carry LIGHT tometastatic tumor that expresses her2/neu and then can generate a localimmune response to clear tumor. Therefore, the fusion protein can bedelivered through any systemic and local route and the fusion proteinwill be more localized to tumors due to the specificity of antibody oranother agent to tumor antigens.

Example 2—Functional Activities of a LIGHT Conjugated Antibody

The ability of antibody-LIGHT to bind to the receptors of LIGHT, LTβRand HVEM, is determined by flow cytometry with LTβR-Ig and HVEM-Ig,respectively. The functional activity of antibody-LIGHT is tested firstin vitro for its ability to costimulate T cells in the presence ofsuboptimal doses of plate-bound anti-CD3. The functionality ofantibody-LIGHT seems comparable with that of anti-CD28.

To test whether Antibody-LIGHT fusion protein inhibits tumor growth invivo, mice are injected s.c. with 5×10⁴ tumor cells for ten days andthen treated with 10 μg of the fusion protein. The inhibition of tumorgrowth is demonstrated with a small dose of fusion protein, i.e. 10 μg,which allows strong immunity against tumor.

This example demonstrates the ability of antibody-LIGHT to bind to thereceptors of LIGHT, LTβR and HVEM by flow cytometry with LTβR-Ig andHVEM-Ig, respectively, and that a tumor specific antibody coupled withLIGHT stimulates immunity to reduce tumor growth.

Example 3—Combination Treatment of Antibody-LIGHT Couple and LocalDelivery of Adenovirus Expressing LIGHT

An important utility of an antibody-LIGHT fusion protein or conjugate isthat such targeting reagents may be very potent to clear small numbersof metastastic tumor cells or residual cancer cells that do noteffectively stimulate the immune system. A combination therapy thatincludes antibody-LIGHT and adenovirus expressing LIGHT, or Ad-LIGHT,are tested.

Tumor cells are inoculated at two sites, one with 10⁶ and the other sidewith 1×10⁴. Two weeks later, the larger tumor (10⁶) is treated withAd-LIGHT and surgically removed two weeks after treatment. Mice aretreated systemically with Antibody-LIGHT at doses described herein. Thismodel determines whether Antibody-LIGHT in combination with localdelivery of Ad-LIGHT to primary tumor is a potent reagent for treatingdistal tumors. 2C T cells, which are readily identified by theclonotypic antibody (1B2), can be adoptively transferred to the tumorbearing mice as a model for tumor antigen-specific CD8⁺ T cells. Thetrafficking, proliferation, and activation of adoptively transferred 2CT cells is monitored and compared with different therapeutic strategies.

Two clinically relevant delivery systems, Ad-LIGHT and Antibody-LIGHT,are expected to effectively target LIGHT to the tumor tissue andsubsequently destroy not only the primary tumors but also distalmetastases. The sustained expression of LIGHT long enough to create aLIGHT-mediated lymphoid-like structure induces the desired anti-tumorCD8⁺ T cell responses.

Example 4—Anti-her2/Neu Antibody-LIGHT Therapy for Breast Cancer

One fifth of breast cancer and colon cancer patients express Her2/neu.Generally, antibody to Her2 slows down the growth of these tumors butdoes not eradicate them. Anti-Her2/neu antibody coupled with LIGHTtargets LIGHT to the site of metastatic tumors. The anti-Her2/neuantibody slows down the growth of tumor and induces apoptosis, whichallows the coupled LIGHT to induce LIGHT-mediated recruiting andactivating of T cells to occur inside tumor. Additionally, LIGHT alsorecruits FcR+ cells to enhance the therapeutic effect of anti-neuantibody. In an experimental model, doses as low as 10 μg of a tumorantibody linked with LIGHT slowed down the growth of tumor in mice.Other lower or higher doses are contemplated. Anti-Her2/neuantibody-LIGHT is a novel treatment for breast cancer metastases. FIG. 2shows that Adv-mmlit inhibits neu+N202 tumor growth.

Example 5—Use of Chemotherapy Drugs in Combination with Antibody-LIGHTFusion or Conjugates

A tumor-specific antibody-LIGHT fusion protein or conjugate is furthercoupled with an anti-tumor agent such as for example, doxorubicin,paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide,5-fluoro uridine, Leucovorin, Irinotecan (CAMPTOSAR™ or CPT-11 orCamptothecin-11 or Campto), Carboplatin, fluorouracil carboplatin,edatrexate, gemcitabine, or vinorelbine or a combination thereof. Thesedrugs can either be administered separately or co-administered byconjugation or coupling with the Antibody-LIGHT fusion protein orconjugate.

This combination therapy may also be co-administered with gene therapywhereby a nucleic acid capable of expressing a protease resistant LIGHTis delivered inside a tumor. Adeno-viral vectors harboring LIGHT nucleicacid sequences, or Ad-LIGHT, are suitable.

Example 6—Synergistic Suppression of Tumors by Anti-her2 Antibody andAd-LIGHT Treatment

The synergy of anti-neu antibody with LIGHT. TUBO. TUBO is a cloned cellline generated from a spontaneous mammary gland tumor from a BALB-neuTmouse and highly expresses HER-2 protein on the cell membrane. Thistumor line is sensitive to anti-neu antibody treatment in vivo and invitro. However, when a tumor is well established, the effect of eitherantibody or LIGHT alone is diminished. After anti-neu antibody isdiscontinued, TUBO cells can recover within 3-4 weeks. To determinewhether there is a synergy between the two, TUBO cells were establishedfor 18 days and then treated with both ad-LIGHT and anti-neu antibodyonce a week for three weeks. Impressively, no tumor can be detected inthis combination while tumor grows progressively with single treatmentof either (FIGS. 4-5). All five mice in each group have tumors, exceptfor those administered the combinational treatment.

Thus combining LIGHT-mediated therapy, e.g., by Ad-LIGHT expressingvector or by another stable LIGHT presentation to tumor cells with anyother anticancer therapy provides a synergistic tumor suppressiontherapeutics.

Example 7—Generation of Antibody-LIGHT Fusion Proteins

To express sc-Fv-LIGHT, scFV-58 LIGHT (LIGHT fragment with amino acidpositions 58-240) and scFV-85 LIGHT (LIGHT fragment with amino acidpositions 85-240, bypassing protease site of 81-84) were constructed.Flag tap was attached to the LIGHT fragment following western blottingsince anti-Flag antibody is very specific and sensitive. Such plasmidswere transfected into a 293 cell line. The cells were harvested one weeklater and lysates were prepared and blotted with anti-flag antibody.Visualization of the anti-Flag western blot shows that the expression ofscFv-85 LIGHT expression is higher than scFv-58 LIGHT.

This demonstrates that the antibody-LIGHT fusion construct generatesfusion proteins and that resulting fusion proteins can be isolated,purified and used to demonstrate that antibody-LIGHT fusion proteinsspecifically targets tumor cells and stimulates production of T-cellsagainst the tumor cells. Similar fusion proteins of LIGHT can be madewith any other antibody that is directed against a tumor cell surfaceantigen and preferably that targets a tumor-specific cell surfaceantigen.

Example 8—Antibody-LIGHT Fusion Constructs

To generate a tumor targeting antibody-LIGHT immunocytokine, thefollowing steps were taken:

-   -   a. LIGHT was engineered for increased stability/affinity;    -   b. a scFV(neu)-LIGHT fusion protein was generated for        production; and    -   c. the scFV(neu)-LIGHT fusion protein was tested in vitro and in        vivo

To engineer LIGHT:

human LIGHT was used as a platform for the engineering.

-   -   Human LIGHT seems more stable (YD and prior expression) than        mouse LIGHT; and    -   Human LIGHT cross-reacts (weakly) to murine receptors.

Criteria for Engineering of LIGHT

-   -   a. Equivalent binding to murine and human LTbR and HVEM, and if        possible, decreased to DcR3; and    -   b. Improved expression/stability to ease production.

Proteins that were engineered using yeast-display include the following:2C T-cell receptor, Ly49C—c-type lectin NKR, 2B4(CD244)-Ig-like NKR,CD48—Ig-like NKR and murine and human KLRG1—c-type lectin NKR.Higher-affinity clones in other CDRs show excellent peptide specificity.(FIGS. 12-13.)

Engineering of LY49C allowed high resolution crystal structure ofLY49C-OVA/K^(b) complex. (FIG. 14.) There is a “domino” effect for theinfluence of peptides on pepMHC binding and specificity. A network ofhydrogen bonding interacts from the peptide down to Ly49C contactregion. (FIG. 15.)

An engineered CD48 facilitated crystallization of the 2B4-CD48 complex.(FIG. 16.)

hLIGHT was engineered using yeast-display. hLIGHT was fused to matingadhesion receptor Aga2 using epitope Tags for normalization.Equilibrium, kinetic and thermal stability analysis was by flowcytometry. (FIGS. 17-19)

The mutants of human LIGHT had improved binding properties and affinitywhen tested against the mouse and human LTβR and HVEM. (FIGS. 20-21.)

A scFV (neu)-LIGHT fusion protein was generated. A glutamine-synthetasevector allowed gene amplification with MSX, and adeno E1aco-transfection was used for transcriptional enhancement. A C-terminalStrep-Tag II sequence provides detection by western blot, ELISA, flowcytometry and high-specificity 1-step purification. (FIG. 22)

A new set of clones was isolated with favorable binding for all 4desired receptors. scFV-LIGHT fusion was produced in CHO cells. BothscGV and LIGHT bound their respective ligands. (FIGS. 23-24)

scFV(neu)-LIGHT fusion protein decreases growth of Tubo cells inculture. Fusion protein mediated cell death was due to the directeffects of LIGHT on LTβR expressed on tumor cells. (FIGS. 25-27)

Materials and Methods

The Generation of Fusion Protein of Antibody-LIGHT.

A recombinant antibody construct designated heterominibody was developedthat allows for the specific targeting of LIGHT to an antibody thatbinds to a tumor antigen or tumor cells with high affinity usingstandard protocol.

Mice, Cell Lines, and Reagents.

Female C3 HXC57BL/6 F1 (C3B6F1) mice, 4-8 weeks old were purchased fromthe National Cancer Institute, Frederick Cancer Research Facility,(Frederick, Md.). C57BL/6-RAG-1-deficient (RAG-1^(−/−)) mice werepurchased from the Jackson Laboratory (Bar Harbor, Me.). H-Y TCRtransgenic mice (H—Y mice) on the RAG-2-deficient/B6 background werepurchased from Taconic Farms (Germantown, N.Y.). 2C TCR transgenic miceon RAG-1-deficient background bred into B6 for 10 generations (2C mice)were provided by J. Chen (Massachusetts Institute of Technology, Boston,Mass.). OT-1 TCR transgenic mice (OT-1 mice) were provided by A. Ma (TheUniversity of Chicago). RAG-1^(−/−), H-Y, 2C, OT-1 mice were bred andmaintained in the specific pathogen-free facility at the University ofChicago. Animal care and use were in accord with institutionalguidelines.

The AG104A expressing murine H-2L^(d) (AG104-L^(d)), the transfectant ofAG104A cells, has been described previously (Wick M, 1997, JEM186:229-38). These tumor cell lines were maintained in DMEM (Mediatech)supplemented with 10% FCS (Sigma-Aldrich), 100 U/ml penicillin, and100·mu·g/ml streptomycin (BioWhittaker). The hybridoma cell linesproducing anti-L^(d) (clone 30-5-7) and anti-2C TCR (1B2) antibodieswere obtained from D. Sachs (National Institutes of Health, Bethesda,Md.) and T. Gajweski (The University of Chicago), respectively.

Monoclonal antibodies produced by hybridomas were purified from theculture supernatant with protein G column by procedures known to thoseof skill in the art. The antecedent 1B2 antibody was conjugated to FITCor biotin by the Monoclonal Antibody Facility of The University ofChicago. PE-coupled anti-CD8 antibody, Cy-chrome (CyC)-coupledstreptavidin, CyC-coupled anti-CD44 antibody, PE-coupled anti-CD62Lantibody and PE-coupled Th1.2 antibody were purchased from BDBiosciences. FITC-conjugated-goat-anti-mouse IgG was purchased fromCaltag. PE-coupled streptavidin was purchased from Immunotech.PE-coupled donkey anti-human IgG was purchased from JacksonImmunological Research Lab (West grove, PA). Biotinylated goat anti-SLCantibody was purchased from R&D systems Inc. (Minneapolis, Minn.). APconjugated rabbit anti-goat Ig antibody was purchased from VectorLaboratories Inc. (Burlingame, Calif.). Purified goat anti-SLC antibodywas purchased from PeproTech (Rock hill, NJ). Collagenase (type 4) waspurchased from Sigma-Aldrich. CFSE was purchased from Molecular Probes.

Tumor Growth In Vivo.

Tumor cells were injected subcutaneously into the lower back, that is,0.5-1 cm above the tail base of the mice. Tumor growth was measuredevery 3 to 4 days with a caliper. Size in cubic centimeters wascalculated by the formula πabc/6, where a, b, and c are three orthogonaldiameters.

Histology.

Tumor tissues for histology examination were collected at time indicatedand fixed in 10% neutral buffered formalin, processed to paraffinembedment, and stained with hematoxylin and eosin. Forimmunohistochemical staining of SLC, tumor tissues were harvested,embedded in OCT compound (Miles-Yeda, Rehovot, Israel) and frozen at−70.degree. C. Frozen sections (5-10 μm thick) were fixed in cold 2%formalin in PBS and permeablized with 0.1% saponin/PBS. The sectionswere preblocked with 5% goat serum in 0.1% saponin/PBS for half an hourat room temperature in a humidified chamber. Staining for SLC was doneby first incubating with biotinylated goat anti-SLC antibody (R&Dsystems Inc. Minneapolis, Minn.) at a 1/25 dilution in blocking buffer.Alkaline phosphatase conjugated rabbit anti-goat Ig antibody (VectorLaboratories Inc. Burlingame, Calif.) was added 2 h later. Forimmunofluorescence staining, sections were blocked with 2% normal mouseserum, rabbit serum, and goat serum in PBS for half an hour at roomtemperature in a humidified chamber. Blocking solution was replaced with50 μl of primary Abs. PE-conjugated anti-Th1.2 (BD PharMingen), orPE-conjugated anti-CD8 (BD PharMingen), diluted 1/100 in blockingsolution, and sections were incubated for 1 h at room temperature in ahumid chamber. Specimens were mounted in Mowiol 4-88 (BD Biosciences, LaJolla, Calif.) containing 10% 1,4-diazobicyclo [2.2.2]octane. Sampleswere analyzed within 48 h using a Zeiss Axioplan microscope (Zeiss,Oberkochen, Germany) and a Photometrics PXL CCD camera (Photometrics,Tucson, Ariz.). No-neighbor deconvolution was performed using Openlabv2.0.6 (Improvision, Lexington, Mass.).

ELISA for CCL21.

Tumor homogenates were prepared and assayed for CCL21. Comparable amountof tumor tissues from tumor-bearing mice were collected and weighed,homogenized in PBS that contained protease inhibitors, and thesupernatants were collected by centrifugation. Polystyrene 96-wellmicrotiter plates (Immulon 4, Dynatech Laboratories, Chantilly, Va.)were coated with goat anti-mouse CCL21 at 2 μg/ml in PBS and were thenblocked with 0.1% bovine serum albumin (BSA) in PBS for 30 min at roomtemperature. After washing, serial dilutions of standards of knownconcentrations (Recombinant CCL21, 50 ng/ml, R&D) and samples were addedand incubated for 2 h at room temperature. After 3 washes, biotinylatedrabbit anti-SLC Ab was added to the wells. After 2 h incubation andwashing, 50 μl of a 1/1000 diluted alkaline phosphatase-conjugatedavidin (Dako) was added for 1 h and then developed. Color developmentwas measured at 405 nm on an automated plate reader (Spectra-Max 340,Molecular Devices, Sunnyvale, Calif.) and The amount of CCL21 wasdetermined by ELISA from the standard curve, and normalized according totissue weight. Data are mean.+−.s.d.

T-Cell Co-Stimulation Assay.

T cells were purified by a negative selection method in the magneticfield as instructed by the manufacture (Miltenyi Biotec, Auburn,Calif.). The purity of isolated T cells was greater than 95%, asassessed by flow cytometry using monoclonal antibody against CD3. Platescoated with 0.2 g/ml monoclonal antibody against CD3 were further coatedat 37°. C. for 4 h with Mutant LIGHT-flag. After being washed, purifiedT cells (1×10⁶ cells/ml) were cultured in the wells. Monoclonal antibodyagainst CD28 (1 μg/ml) was used in soluble form. In all assays, theproliferation of T cells was assessed by the addition of 1Ci/well³H-thymidine during the last 15 h of the 3-dayculture³H-thymidine incorporation was measured in a TopCount microplatescintillation counter (Packard instrument, Meriden, Conn.).

Cell Isolation from Tumor Tissue.

The mice were first bled to decrease the blood contamination of tumortissue. The tumor tissues were collected, washed in the PBS, cut intopieces, and resuspended in DMEM supplemented with 2% FCS and 1.25 mg/mlcollagenase D (collagenase D solution) for 40 min in a 37.degree. C.shaking incubator. The single cell suspension was collected after 40min, and the cell clumps were digested for another 40 min in thecollagenase D solution until all tumor tissue had resolved into a singlecell suspension.

Pharmaceutical Compositions.

Therapeutic compositions used herein can be formulated intopharmaceutical compositions comprising a carrier suitable for thedesired delivery method. Suitable carriers include materials that whencombined with the therapeutic composition retain the anti-tumor functionof the therapeutic composition. Examples include a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like. Therapeutic formulationscan be solubilized and administered via any route suitable to deliverthe therapeutic composition to the tumor site. Potentially effectiveroutes of administration include intravenous, parenteral,intraperitoneal, intramuscular, intratumor, intradermal, intraorgan,orthotopic, and the like. A formulation for intravenous injectionincludes the therapeutic composition in a solution of preservedbacteriostatic water, sterile unpreserved water, and/or diluted inpolyvinylchloride or polyethylene bags containing sterile sodiumchloride for injection. Therapeutic protein preparations can belyophilized and stored as sterile powders, preferably under vacuum, andthen reconstituted in bacteriostatic water (containing for example,benzyl alcohol preservative) or in sterile water prior to injection.Dosages and administration protocols for the treatment of cancers usingthe methods disclosed herein may vary with the method and the targetcancer, and generally depend on a number of factors appreciated andunderstood in the art.

Measurement of Cytokines in the Spleen and Tumor.

Tumor and spleen homogenates was prepared as described (Yu et al., 2003JEM197:985-995). Briefly, comparable amounts of tumor or spleen tissueswere collected, weighed and homogenized in PBS containing proteaseinhibitors, and the supernatants were collected by centrifugation. Theamount of cytokines in the supernatants was quantified using thecytometric bead array kit (CBA) (BD Biosciences) on a FACS Calibercytometer equipped with CellQuestPro and CBA software (Becton Dickinson)according to manufacturer's instruction.

Statistical Analysis for Difference in Tumor Growth.

Because the tumor growth was observed repeatedly over time on the samemouse, the random effect models for longitudinal data were used toanalyze such data. For each experiment, the tumor growth was assumed todepend on treatment and to follow a linear growth rate over time. Themodel gave an overall estimate of the intercept and slope of the lineargrowth for each group. Both the intercept and slope were allowed to varyamong individual mouse. The slope, i.e., the growth rate was comparedwas different among different treatment groups. Because the actual tumorgrowth may not follow a linear growth trend over the entire follow upperiod. The increase of tumor growth was slow at the early stage andbecame rapid at the later stage in some experiments. A quadratic termwas added to the follow-up time in the above random effect models.

Generation of mutant LIGHT Expression Vectors and Clones pcDNA3.1-LIGHTwas used as template to generate two dsDNA fragments A and B by PCR. Forgeneration of fragment A (about 500 b.p.), sense primer5′-CATGGATCCAAGACCATGGAGAGTGTGGTACA-3′ (SEQ ID NO: 36) (the bold textindicated BamHI site) and antisense primer5′-AGATCGTTGATCTTGCCAGGAGCCTTTGCC-3′ (SEQ ID NO: 37) were used. Togenerate fragment B (about 200 b.p.), sense primer5′-GGCAAAGGCTCCTGGCAAGATCAACGATCT-3′ (SEQ ID NO: 38) and antisenseprimer 5′-ACCTCTAGATCAGACCATGAAAGCTCCGA-3′ (SEQ ID NO: 39) (theunderlined text indicated XbaI site) were used. The antisense primer forfragment A is complimentary with sense primer for fragment B, whichcovers sequences for amino acid (a.a.) 73-87 among which a.a. 79-82 weredeleted. Fragments A and B were mixed, denatured at 94 degrees C. andcooled down to room temperature to anneal the two DNA fragments. Theannealed DNA product was used as template for a PCR reaction and theproduct was cloned into pcDNA3.1 using BamHI and XbaI. The deletion ofa.a. 79-82 was verified by sequencing. To generate pMFG-mutant LIGHT,pcDNA3.1-mutant LIGHT was digested with NcoI and BamHI and ligated to aNcoI and BamHI-digested the pMFG-S-TPA plasmid (Mulligan R C,Massachusetts Institute of Technology, Boston, Mass.).

Delivery of a nucleic acid encoding mutant human LIGHT into a patientmay be either direct, in which case the patient is directly exposed tothe nucleic acid or nucleic acid-carrying vectors, or indirect, in whichcase, tumor cells obtained from a biopsy are first transformed with thenucleic acids in vitro, irradiated and then transplanted into thepatient. These approaches are routinely practiced in gene therapies forsuppressing tumors or treating other illness.

Delivery of Nucleic Acids.

The nucleic acid sequences are directly administered in vivo, where theyare expressed to produce the encoded products. This can be accomplishedby any of numerous methods known in the art, e.g., by constructing themas part of an appropriate nucleic acid expression vector andadministering it so that they become intracellular, e.g., by infectionusing defective or attenuated retroviral or other viral vectors (U.S.Pat. No. 4,980,286), or by direct injection of naked DNA, or by use ofmicroparticle bombardment, or coating with lipids or cell-surfacereceptors or transfecting agents, encapsulation in liposomes,microparticles, or microcapsules, or by administering them in linkage toa peptide which is known to enter the nucleus, by administering it inlinkage to a ligand subject to receptor-mediated endocytosis (which canbe used to target cell types specifically expressing the receptors),etc. Alternatively, the nucleic acid can be introduced intracellularlyand incorporated within host cell DNA for expression, by homologousrecombination.

Biodegradable microspheres have also been used in gene delivery thatencapsulate the nucleic acid. Microspheres such as matrices, films, gelsand hydrogels which include hyaluronic acid (HA) derivatized with adihydrazide and crosslinked to a nucleic acid forming slow releasemicrospheres have been used to deliver nucleic acids. U.S. Pat. No.6,048,551 discloses a controlled release gene delivery system utilizingpoly (lactide-co-glycolide) (PLGA), hydroxypropylmethyl cellulosephthalate, cellulose acetate phthalate, and copolymer microspheres toencapsulate the gene vector.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude materials that when combined with the therapeutic compositionretain the anti-tumor function of the therapeutic composition. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like. Therapeutic formulationscan be solubilized and administered via any route capable of deliveringthe therapeutic composition to the tumor site. Potentially effectiveroutes of administration include, but are not limited to, intravenous,parenteral, intraperitoneal, intramuscular, intratumor, intradermal,intraorgan, orthotopic, and the like. A preferred formulation forintravenous injection comprises the therapeutic composition in asolution of preserved bacteriostatic water, sterile unpreserved water,and/or diluted in polyvinylchloride or polyethylene bags containingsterile sodium chloride for injection. Therapeutic protein preparationscan be lyophilized and stored as sterile powders, preferably undervacuum, and then reconstituted in bacteriostatic water (containing forexample, benzyl alcohol preservative) or in sterile water prior toinjection. Dosages and administration protocols for the treatment ofcancers using the foregoing methods will vary with the method and thetarget cancer, and will generally depend on a number of other factorsappreciated in the art.

Delivery Using Viral Vectors.

Viral vectors that contain nucleic acid sequences encoding an antibodyof the invention are used for delivering specific nucleic acids. Forexample, a retroviral vector can be used. These retroviral vectorscontain the components necessary for the correct packaging of the viralgenome and integration into the host cell DNA. The nucleic acidsequences encoding the desired protein to be used in gene therapy arecloned into one or more vectors, which facilitates delivery of the geneinto a patient. Adenoviruses are other viral vectors that can be used ingene therapy. Adenoviruses are especially attractive vehicles fordelivering genes to respiratory epithelia and other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Adeno-associated virus (AAV)has also been proposed for use in gene therapy (U.S. Pat. No.5,436,146). Lentiviruses are promising for use in gene therapy.

Transfecting cells in tissue culture followed by delivery to patients.Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient. In this method, the nucleic acidis introduced into a cell prior to administration in vivo of theresulting recombinant cell. Such introduction can be carried out by anymethod known in the art, including but not limited to transfection,electroporation, microinjection, infection with a viral or bacteriophagevector containing the nucleic acid sequences, cell fusion,chromosome-mediated gene transfer, microcell-mediated gene transfer,spheroplast fusion, etc. The technique should provide for the stabletransfer of the nucleic acid to the cell, so that the nucleic acid isexpressible by the cell and preferably heritable and expressible by itscell progeny.

The resulting recombinant cells may be irradiated and can be deliveredto a patient by various methods known in the art. Recombinant cells(e.g., hematopoietic stem or progenitor cells) are preferablyadministered intravenously. The amount of cells envisioned for usedepends on the desired effect, patient state, etc., and can bedetermined by one skilled in the art. Cells into which a nucleic acidcan be introduced for purposes of gene therapy encompass any desired,available cell type, and include but are not limited to epithelialcells, endothelial cells, keratinocytes, fibroblasts, muscle cells,hepatocytes, blood cells such as T lymphocytes, B lymphocytes,monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,granulocytes; various stem or progenitor cells, in particularhematopoietic stem or progenitor cells, e.g., as obtained from bonemarrow, umbilical cord blood, peripheral blood, fetal liver, etc.

Vaccines.

As used herein, the term “vaccine” refers to a composition (e.g., amutant human LIGHT antigen and an adjuvant) that elicits atumor-specific immune response. These vaccines include prophylactic(preventing new tumors) and therapeutic (eradicating parental tumors). Avaccine vector such as a DNA vaccine encoding a mutant human LIGHT canbe used to elicit immune response against tumors. The response iselicited from the subject's own immune system by administering thevaccine composition at a site (e.g., a site distant from the tumor). Theimmune response may result in the eradication of tumor cells in the body(e.g., both primary and metastatic tumor cells). Methods for generatingtumor vaccines are well known in the art (See e.g., U.S. Pat. Nos.5,994,523 and 6,207,147 each of which is herein incorporated byreference).

The vaccines may comprise one or more tumor antigens in a pharmaceuticalcomposition. In some cases, the tumor antigen is inactivated prior toadministration. In other embodiments, the vaccine further comprises oneor more additional therapeutic agents (e.g., cytokines or cytokineexpressing cells).

In certain cases, cells selected from a patient, such as fibroblasts,obtained, for example, from a routine skin biopsy, are geneticallymodified to express one or of the desired protein. Alternatively,patient cells that may normally serve as antigen presenting cells in theimmune system such as macrophages, monocytes, and lymphocytes may alsobe genetically modified to express one or more of the desired antigens.The antigen expressing cells are then mixed with the patient's tumorcells (e.g., a tumor antigen), for example in the form of irradiatedtumor cells, or alternatively in the form of purified natural orrecombinant tumor antigen, and employed in immunizations, for examplesubcutaneously, to induce systemic anti-tumor immunity. The vaccines maybe administered using any suitable method, including but not limited to,those described above.

Cancer metastasis may be reduced by stimulation of at least one of thefollowing including chemokines, adhesion molecules, and costimulatorymolecules for priming naive T-cells. Cancer types include breast cancer,lung cancer, prostate cancer, colon cancer, and skin cancer.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe performed following the method of Winter and co-workers [Jones etal., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Various forms of a humanized antibody-LIGHT fusions or conjugates arecontemplated. For example, the humanized antibody may be an antibodyfragment, such as a Fab, which is conjugated with LIGHT or anextracellular fragment thereof. Alternatively, the humanized antibodymay be an intact antibody, such as an intact IgG1 antibody.

As an alternative to humanization, human antibodies can be generated.For example, it is possible to produce transgenic animals (e.g., mice)that are capable, upon immunization, of producing a variety of humanantibodies in the absence of endogenous immunoglobulin production. See,e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993).

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle (See e.g., Johnson, Kevin S, and Chiswell, David J, CurrentOpinion in Structural Biology 3:564-571 (1993)). Human antibodies mayalso be generated by in vitro activated B cells (see U.S. Pat. Nos.5,567,610 and 5,229,275).

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies. However, these fragments can also beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage libraries.The antibody fragment may also be a “linear antibody”, e.g., asdescribed in U.S. Pat. No. 5,641,870 for example. Such linear antibodyfragments may be monospecific or bispecific.

Conjugates of the antibody and a co-stimulatory molecules such as LIGHTmay be made using a variety of bifunctional protein coupling agents suchas N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediaminc), di isocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). An extracellular domain of LIGHT orfragments thereof are conjugated to an antibody or antibody fragmentsthat are specific to a tumor antigen, preferably, a surface tumorantigen.

Alternatively, a fusion protein comprising the anti-tumor antigenantibody and LIGHT may be made, e.g., by recombinant techniques orpeptide synthesis. The length of DNA may comprise respective regionsencoding the two portions of the conjugate either adjacent one anotheror separated by a region encoding a linker peptide which does notdestroy the desired properties of the conjugate.

The antibody-LIGHT complexes disclosed herein may also be formulated asimmunoliposomes. A “liposome” is a small vesicle composed of varioustypes of lipids, phospholipids and/or surfactant which is useful fordelivery of a drug to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes. Liposomes containing the antibodyare prepared by methods known in the art, such as described in U.S. Pat.Nos. 4,485,045 and 4,544,545; and WO97/38731 published Oct. 23, 1997.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

For the prevention or treatment of disease, the dosage and mode ofadministration will be chosen by the physician according to knowncriteria. The appropriate dosage of LIGHT, Antibody-LIGHT conjugate orfusion product may depend on the type of cancer to be treated, theseverity and course of the disease, the size of the tumor, the extent ofmetastases, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The LIGHT or antibody-LIGHT composition is suitablyadministered to the patient at one time or over a series of treatments.Preferably, the composition is administered by intravenous infusion orby subcutaneous injections. Depending on the type and severity of thedisease, about 1·mu·g/kg to about 50 mg/kg body weight (e.g., about0.1-15 mg/kg/dose) of antibody can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A dosing regimenmay include administering an initial loading dose of about 5 mg/kg,followed by a weekly maintenance dose of about 2 mg/kg of the anti-TATantibody. However, other dosage regimens may be useful. A typical dailydosage might range from about 1·mu·g/kg to 100 mg/kg or more, dependingon the factors mentioned above. For repeated administrations overseveral days or longer, depending on the condition, the treatment issustained until a desired suppression of disease symptoms occurs, e.g.,reduction in tumor size/volume and reduction in metastases. The progressof this therapy can be monitored by conventional methods and assays andbased on criteria known to the physician or other persons of skill inthe art.

TABLE I Ad-LIGHT eradicates metastases and promotes long-term survivalTime of Number of Mice Free Sacrifice, of Tumor Cells in the Treatmentsand Time^(a) In Days^(a) Lung/All Mice (%)^(b) None 14   3/22 (13.6%)Surgery on day 14 35 0/10 (0%) Ad-control^(c) on day 14 + 35 0/35 (0%)Surgery on day 24 Ad-LIGHT^(c) on day 14 + 35  18/35 (51.4%) Surgery onday 24 Ad-LIGHT and CD8 35 0/35 (0%) depletion^(d) on day 14 + Surgeryon day 24 ^(a)Days after primary tumor inoculation. ^(b)Pooled fromseveral independent experiments. ^(c)Total of 2.5 × 10⁹ PFU Ad-control(LacZ) or Ad-LIGHT was injected intratumorly per mouse. ^(d)A total of125 mg of depleting anti-CD8 Ab was injected on day 14 and once everyweek.

The invention claimed is:
 1. A method of reducing the growth of primarytumor or cancer metastasis, the method comprising: (a) administering apharmaceutical composition comprising a mutant human LIGHT moleculecomprising an amino acid sequence selected from the group consisting ofSEQ ID Nos: 3, 4, 5, and 20, wherein the composition is sufficient tostimulate of cytotoxic T lymphocytes against tumor cells.
 2. The methodof claim 1, wherein the pharmaceutical composition is administeredintravenously.
 3. The method of claim 1, wherein the cancer metastasisis reduced by stimulating production of at least one of chemokines,adhesion molecules, and costimulatory molecules for priming naiveT-cells.
 4. The method of claim 1, wherein the cancer is selected fromthe group consisting of breast cancer, lung cancer, prostate cancer,colon cancer, renal cancer, liver cancer, leukemia, and skin cancer. 5.The method of claim 1 further comprising administering achemotherapeutic agent or radiotherapy.
 6. The method of claim 1,wherein the mutant human LIGHT molecule is linked to a tumor-specificagent.
 7. The method of claim 6, wherein the agent is an antibody thatrecognizes a surface tumor antigen.
 8. The method of claim 6, whereinthe agent is an antibody specific to a tumor antigen selected from thegroup consisting of HER2, HER4, HERB, EGFR, STEAP, c-Met,alphafetoprotein (AFP), Carcinoembryonic antigen (CEA), CA-125, MUC-1,abnormal products of ras or p53, and DcR3.
 9. The method of claim 6,wherein the agent is an antibody conjugated to the LIGHT fragmentchemically or fused to the LIGHT fragment recombinantly.